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REVIEW

Natural products and Chagas disease: a review of plant compounds studied for activity against Trypanosoma cruzi
Erika Izumi,a T^nia Ueda-Nakamura,b Benedito Prado Dias Filho,ab Valdir Flor^ncio Veiga Jniorc a e u and Celso Vataru Nakamura*ab
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Received 31st May 2010 DOI: 10.1039/c0np00069h

Covering: 1995 to 2010 Here, we review studies that have investigated the activity of plant-derived compounds against Trypanosoma cruzi, the etiologic agent of Chagas disease. In the last decade, more than 300 species belonging to almost 100 families have been evaluated for activity, and here we describe the compounds isolated; 85 references are cited.

1 2 3 4 5

Introduction Activities of natural products against Trypanosoma cruzi Opportunities for future research Acknowledgements References

1 Introduction
Natural products are an increasing source of new drugs that may, in the near future, replace current medications that have severe side-effects. Natural products with antibacterial, antiprotozoal, antimycobacterial, antileishmanial, antitumor, and anti-HIV15 properties have been identied in recent decades. Ancient customs have in many cases led to modern plant research because, for centuries, plant extracts were the only known medicines available. However, with advances in technology, large numbers of natural compounds from animals, marine organisms, and free-living or symbiotic microorganisms have been investigated for new drugs.6,7 American trypanosomiasis, also known as Chagas disease, is endemic in Latin America, where the World Health Organization estimates that about 15 million people are infected and that almost 30 million live in risk areas.8 The disease was rst
Programa de Ps-Graduaca em Microbiologia, Universidade Estadual de o o Londrina, Rodovia Celso Garcia Cid s/n, 86051-990 Londrina-PR, Brazil. E-mail: cvnakamura@uem.br; Fax: +55 44 3011-5050; Tel: +55 44 30415012 b Departamento de Ci^ncias Bsicas da Sade, Laboratrio de Inovaca e a u o o Tecnolgica no Desenvolvimento de Frmacos e Cosmticos, o a e Universidade Estadual de Maring, Av. Colombo 5790, 87020-900 a Maring-PR, Brazil a c Departamento de Qumica, Instituto de Ci^ncias Exatas, Universidade e Federal do Amazonas, Av. Gal. Rodrigo Octvio Jorda Ramos 3000, a o Japiim, 69077-000 Manaus, Brazil
a

described in 1909 by the Brazilian physician Carlos Chagas, who identied the symptoms and described both the vectors and the life cycle of the parasite, Trypanosoma cruzi.9 Although Chagas disease has been recognised for more than a century, paleobiology experiments detected the presence of the protozoan in human populations from 9000 years ago, specically in tissues collected from mummies in the Andean region.10,11 T. cruzi is a parasitic agellate protozoan that belongs to the order Kinetoplastida and the family Trypanosomatidae, along with Leishmania and T. brucei, which are also human parasites of large impact on human health. In its natural cycle, T. cruzi is transmitted to wild mammals, its vertebrate hosts, by hematophagous Triatominae insects. The proximity of human populations to natural habitats caused another parasite cycle to arise the domestic one, in which the vertebrate hosts are humans and their domestic animals.12 T. cruzi is known to have the following three main morphological forms during its life cycle: the epimastigote (replicative, noninfective), which is found in the midgut of the invertebrate vector; the trypomastigote (nonreplicative, infective), which is the form of the parasite found in both hosts; and the amastigote (replicative, infective), which is the intracellular form found only in mammals, and the most difcult to reach by drugs.13,14 The few symptoms apparent at the beginning of an infection with T. cruzi include fever, malaise, and pain, which can be diagnosed as many other infectious diseases such as dengue or inuenza. Constipation, as well as chest pain and fatigue, when manifested in people of advanced age, may be associated with aging problems and not with the symptoms of the late stage of Chagas disease. The asymptomatic phase of the disease can persist for decades before the rst manifestations begin, or it can last for the hosts entire lifetime, which occurs in the majority of cases.15 This asymptomatic situation may be one explanation for the difculty in nding medicinal plants used popularly to treat
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Chagas disease. In such cases, plants used for other protozoal diseases, especially those with taxonomic similarities, may show better results.16 Socioeconomic factors in countries where the disease is endemic inuence the level of funding for research and development that pharmaceutical companies will dedicate to nding new drugs for neglected diseases, because most infected people live on less than US$2 per day and cannot pay for expensive treatments. Moreover, the long chronic-asymptomatic phase makes it difcult to accurately estimate the number of sufferers, which is higher than ofcial data show.17
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Erika Izumi

Erika Izumi obtained her BSc degree in Biological Sciences in 2004 from the Universidade Estadual de Londrina, Brazil. Currently she is a microbiology PhD student under the supervision of Professor Nakamura at the same university, where she is studying the biological activity of medicinal plants from the Amazon rainforest against protozoan parasites. Her research interests are natural products and their application to neglected diseases. T^nia a Ueda-Nakamura obtained her BSc degree in Pharmacy and Biochemistry in 1980 from the Universidade Estadual de Maring, Brazil. In a 2001 she received her PhD degree in Biophysics from the Universidade Federal do Rio de Janeiro, and currently she is an Associate Professor of the Universidade Estadual de Maring. a Her research interests are antiviral and antiprotozoal activities of natural products. Benedito Prado Dias Filho received his BSc degree in Pharmacy and Biochemistry in 1973 from the Universidade Estadual de Ponta Grossa, Brazil, and in 1992 he obtained his PhD degree in Microbiology from the Universidade Federal do Rio de Janeiro. Currently he is a Full Professor of the Universidade Estadual de Maring and Pro-rector of the a Postgraduate Program and Research. His main interests are nanotechnology and antimicrobial activity of natural products, focusing on dermatophytes.

In the 1970s, nifurtimox and benznidazole were synthesised and belong to the nitrofurans and nitroimidazols, respectively. These drugs are highly toxic to mammalian cells. In Brazil, benznidazole is the only available drug, and its action results in a cure rate of approximately 7080% in the acute phase but only 1020% for chronic infection.18,19 The toxicity manifests in severe side-effects, and many patients abandon the treatment. Even after decades of research there still are no compounds able to cure all Chagas disease patients, and no substitute for the two drugs has been developed.20 In attempts to nd new therapies, synthetic drugs have been designed and evaluated, either alone or in combination with other drugs, against this parasite.2123 The advantage of the synthetic drugs is that, because of the known chemical structures of benznidazole and nifurtimox, alterations of their basic structural frameworks or the construction of new molecules with similar functional groups can guarantee high activity.24,25 Discovery of new molecules from natural sources, however, is much more difcult. In the case of malaria, for example, quinine, an active compound against Plasmodium sp., was isolated from Cinchona sp. bark in the 17th century. Until recently, quinine was the only natural product with adequate activity to prevent and treat the disease and from which synthesised derivatives became commercially successful. The isolation of artemisinin from

Valdir Flor^ncio Veiga Jnior e u

Valdir Flor^ncio Veiga Jnior e u obtained his BSc degree in Chemical Engineering in 1995 and the PhD degree in Chemistry in 2004 from the Universidade Federal do Rio de Janeiro, Brazil. Currently he is an Associate Professor of the Universidade Federal do Amazonas. His research interests are the chemistry of natural products, focusing on medicinal plants of the families Fabaceae, Burseraceae, and Lauraceae.

T^nia Ueda-Nakamura a

Benedito Prado Dias Filho

Celso Vataru Nakamura graduated in Pharmacy and Biochemistry at the Universidade Estadual de Maring, a Brazil, in 1979. He obtained his PhD degree in Microbiology at the Universidade Federal do Rio de Janeiro in 1992, and he undertook postdoctoral research on Biophysics, working with ultrastructural analysis of cellular organelles from parasitic protozoa. Currently he is an Celso Vataru Nakamura Associate Professor at the Universidade Estadual de Maring, a and his research interests are the development of new drugs from natural sources, focusing on antimicrobial, antiprotozoal, and melanogenic activities.
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Artemisia annua, however, has improved the prospects for treatment of quinine-derivative-resistant malarial parasites.2628 The search for natural compounds extracted from plants has intensied in previous years, and the results are promising. The possibility of nding an active molecule with low toxicity is increasing as more plant species are screened. However, the question remains as to whether plants are being screened in the right way to reveal their optimum activity. The efciency of the available drugs and of the new drugs tested also depends on the strain of the parasite.29 Some strains are more resistant to the commonly used drugs, and this difference should be carefully analysed, because it may result from different mechanisms of action and defense. A few studies have used more than a single strain, and this comparison could be important to improve the evaluation of the effects of new extracts and compounds.30 The similarities and differences in the effects of new drugs on different strains can provide new and useful information.

2 Activities of natural products against Trypanosoma cruzi

There are many incentives for studying the medicinal benets of natural products. For Chagas disease, the prospect of developing new drugs has led over the last 15 years to the screening of almost 400 species belonging to more than 100 plant families for activity against T. cruzi. Many extraction processes used only one type of solvent and a single part of the plant; however, others have extracted compounds from various plant parts and used different solvents. Usually, the plant part subjected to the extraction process is the same that is used traditionally, but other parts may be assessed to determine which part contains the highest concentration of the active compounds. In screening for activity, solvents of different polarity should be used for the extractions because it is not certain that only the most polar compounds will show the highest activity. Among the various studies, the following 18 plant species (listed by family, genus, and species) were evaluated by different investigators against different life stages of T. cruzi: Anacardiaceae, Schinus molle;31,32 Annonaceae, Annona muricata,33,34 A. reticulata;33,35,36 Asteraceae, Mikania cordifolia,36,37 Neurolaena lobata,35,36,38 Tagetes lucida,35,36 Tanacetum parthenium,33,39 Tridax procumbens;35,36,38,40 Euphorbiaceae, Croton guatemalensis;35,36 Fabaceae, Gliricidia sepium;35,36,38 Lamiaceae, Marrubium vulgare;32,33 Malpighiaceae, Byrsonima crassifolia;35,36,38 Myrtaceae, Psidium guajava;33,36 Phytolaccaceae, Petiveria alliacea;35,38 Poaceae, Cymbopogon citratus;40,41 Polypodiaceae, Phlebodium aureum;33,36 Smilacaceae, Smilax lundelli;35,36 and Sterculiaceae, Chiranthodendron pentadactylon.33,36 The plant part used and the solvent chosen often differ. Even with the same extraction process, the results obtained were not the same, principally because of the different methodologies employed. Promising results were obtained upon exposing the parasite to some plant extracts. Hexane extracts of Polygala sabulosa and aqueous extracts of P. cyparissias (Polygalaceae) showed 50% inhibition of epimastigote growth after 72 h of treatment at concentrations of 1 and 2 mg mL1, respectively.31 For trypomastigotes, Piptadenia africana (Mimosaceae) methanolic
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extract caused lysis in 50% of the parasites at 4 mg mL1 after 96 h,40 and a methanolic extract from Gardenia lutea (Rubiaceae) also promoted the same effect at approximately 22 mg mL1 after 72 h.42 Against amastigotes, a surprising inhibition of 50% at less than 0.25 mg mL1 after 7 days of treatment was detected using methanolic extracts from the following 8 species: Hypoestes forsskalii (Acanthaceae), Kleinia odora and Psiadia punctulata (Asteraceae), Capparis spinosa (Capparidaceae), Euphorbia schimperiana and Ricinus communis (Euphorbiaceae), Marrubium vulgare (Lamiaceae), and Solanum villosum (Solanaceae).32 All of these interesting results could possibly lead to the discovery of new active compounds, especially against intracellular forms of the parasite. Despite the screening of hundreds of species, the major, possibly active compound was only isolated, identied, and evaluated for antiparasitic activity in approximately 10% of the cases. The total of 136 compounds tested against T. cruzi in the last decade are described in Table 1. They are organised by family and species, and the methodology utilised to identify them are indicated in order to compare all reported activities. Due to the great variety of methods used to carry out antiparasitic studies, it is not a simple matter to capture data and determine which method yields the best results and should be continued, or which used as the standard for evaluation. All life stages of the parasite have their own appropriate culture medium (e.g., LIT or BHI). The host cell type chosen for the amastigote and trypomastigote forms also can be different, as can the media used to culture them, for example, DMEM or RPMI 1640. Some data are described precisely, whereas others are given as a range of concentrations, which makes it impossible to discern the true value. These hidden values prevent comparison with other values, and hence hinder discussion and analysis of the activity. The majority of the processes used isolated alkaloids (136), which compose almost 29% of the compounds listed. All compounds isolated from members of the families Ancistrocladaceae, Annonaceae, Rutaceae and Solanaceae, and some from Lauraceae, are alkaloids. Against epimastigotes, only a steroidal alkaloid 36 was tested, resulting in growth inhibition of 50% at 10 mg mL1 after 48 h. For investigation of amastigote survival, isoquinoline alkaloids from the family Ancistrocladaceae were identied. The most active were compounds 6, 8, and 9, which all reduced intracellular replication at concentrations varying from 1.5 to 2.35 mg mL1. Similar activities were seen for 1 and 4, with IC50 values of approximately 39 and 30 mg mL1, respectively, demonstrating that a methyl group bound to nitrogen causes a reduction of almost 10 mg mL1 in IC50. Compounds 2, 7, and 10 also had similar activities at approximately 17 mg mL1. Comparing 7 and 10, it is clear that changes in the position of the methyl and the substitution of a methoxy for a hydroxy did not interfere with activity, as was also observed for the presence of a methyl bound to nitrogen in compound 2. Aporphine alkaloids evaluated against trypomastigotes did not show high antiparasitic activity, except for 11, which caused the death of half the parasite population at 9.32 mM after 24 h. An oxide bound to positive nitrogen could be responsible for the loss of activity of 12, while the absence of a dioxole ring and a hydroxy on 15 could explain its loss of activity. Other alkaloids
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Table 1 Isolated compounds from plants evaluated against Trypanosoma cruzi. Activity in T. cruzia Species Ancistrocladaceae Ancistrocladus congolensis Compounds Epimastigotes Trypomastigotes Intracellular amastigotes 39.9b**** 17.4b**** >90b**** 30.1b**** 14.5b**** 2.35b**** 17.6b**** 1.7b**** 1.5b**** 17.8b**** NT NT NT NT NT 7.6b**** 6.8b**** 33.1b**** >90b**** 18.05b**** 5.72b**** >30b**** NAb*** NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT 93j** NT NT NT NT 7.9b**** 0.2b**** 0.15b**** Ref.

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Ancistrocladus ealaensis Ancistrocladus tanzaniensis Annonaceae Duguetia furfuracea

Ancistrocongoline A (1) Ancistrocongoline B (2) Ancistrocongoline C (3) Ancistrocongoline D (4) Korupensamine A (5) Ancistroealaine A (6) Ancistroealaine B (7) Ancistrotanzanine A (8) Ancistrotanzanine B (9) Ancistrotectoriline A (10) Duguetine (11) Duguetine-b-N-oxide (12) Dicentrinone (13) N-Methyltetrahydropalmatine (14) N-Methylglaucine (15) Knipholone (114) 40 -O-Demethylknipholone-40 -O-b-D-glucoside (115) Gaboroquinone A (116) Gaboroquinone B (117) Anthecotulide (90) 4-Hydroxyanthecotulide (91) 4-Acetoxyanthecotulide (92) 5,6,7-Trihydroxy-40 -methoxyavanone (37) Centratherin (93) Galangin 3-methyl ether (41) Lychnopholide (94) Pinobanksin 3-acetate (38) 15-Deoxygoyazensolide (95) Pinobanksin (39) Galangin (42) Goyazensolide (96) Tectochrysin (43) Luteolin (44) Vicenin-2 (48) Caffeic acid (129) Pinocembrin (40) Isorhamnetin 3-O-glucoside (46) 4,5-di-O-E-Caffeyolquinic acid (130) Quercetin-3-methyl ether (45) Isorhamnetin 3-O-(60 -p-coumaroyl) glucoside (47) Parthenolide (97) Helenalin (98) Mexicanin (99)

NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT 0.5b**** 1.9c**** 3.8c****

NT NT NT NT NT NT NT NT NT NT 9.32c* 30.79c* 18.83c* 9072c* 4957c* NT NT NT NT NT NT NT 20.39b* 52.60d* 10.17d* 66.67d* 16.67d* 65.21d* 35.09d* 13.43d* 98.88d* 42.30d* 38.46d* 41.82d* 15.94d* 40.13d* 18.44d* 74.77d* 32.10d* 17.15d* NT NT NT 62.75d* 56.64d* NT NT NT

43 43 43 43 43 44 44 45 45 45 46 46 46 46 46 47 47 47 47 48 48 48 49 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 39 51 51 52 52 53 53 53

Asphodelaceae Bulbine frutescens

Asteraceae Anthemis auriculata Baccharis retusa Lychnophora pohlii (leaves and owers)

Tanacetum parthenium (aerial parts) Gaillardia megapotamica (aerial parts) Mikania stipulacea Mikania hoehnei Araliaceae Cussonia zimmermannii

ent-9a-Hydroxy-15b-E-cinnamoyloxy-16-kauren-19-oic acid NT (76) 8b-Hydroxyzaluzanin D (102) NT 8-Hydroxyheptadeca-4,6-diyn-3-yl ethanoate (120) 8-Hydroxyheptadeca-1-ene-4,6-diyn-3-yl ethanoate (121) 16-Acetoxy-11-hydroxyoctadeca-17-ene-12,14-diynyl ethanoate (122) Ursolic acid (106) Oleanolic acid (107) Alpinetine (49) NT NT NT

Bignoniaceae Arrabidaea triplinervia (leaves)

NT NT NT

400f* 1600f* NAf*

NT NT NT

54 54 54

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Table 1 (Contd. ) Activity in T. cruzia Species Compounds Epimastigotes Trypomastigotes Intracellular amastigotes Ref.

Clusiaceae Garcinia livingstonei

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6,11-Dihydroxy-3-methyl-3-(4-methylpent-3-enyl)pyrano[2,3- NT c]xanthen-7(3H)-one (109) 4[(E)-3,7-Dimethylocta-2,6-dienyl]-1,3,5-trihydroxy-9HNT xanthen-9-one (110) 1,4,5-Trihydroxy-3-(3-methylbut-2-enyl)-9H-xanthen-9-one NT (111) Garcilivin A (112) NT Garcilivin C (113) NT ent-Naringeninyl-(I-3R,II-8)-4-O-methylnaringenin (50) NT (+)-Volkensiavone (51) NT (+)-Morelloavone (52) NT Biochanin A (53) Geranylgeraniol (77) 18-Hydroxycassan-13,15-diene (78) 6b,18-Dihydroxycassan-13,15-diene (79) 6b-Hydroxy-18-acetoxycassan-13,15-diene (80) 18-Acetoxy-13,15-diene-19-cassanoic acid (81) 6b,13b-Dihydroxy-18-acetoxycassan-14(17),15-diene (82) Citral (103) Cyclocoulterone (83) Komaroviquinone (84) Dracocephalone A (85) 5-epi-Icetexone (86) Linalool (104) Dehydrocostus lactone (100) Zaluzanin D (101) (1R,4S)-1-Hydroperoxy-p-menth-2-en-8-ol acetate (105) Machilin G (62) Galgravin (63) Nectandrin A (64) Nectandrin B (65) Calopiptin (66) Aristolignin (67) Ganschisandrine (68) 4-Acetoxy-1,2-dihydroxyheptadec-16-ene (123) 1-Acetoxy-2,4-dihydroxyheptadec-16-ene (124) 1,2,4-Trihydroxyheptadec-16-ene (125) 4-Acetoxy-1,2-dihydroxyheptadec-16-yne (126) 1-Acetoxy-2,4-dihydroxyheptadec-16-yne (127) 1,2,4-Trihydroxyheptadec-16-yne (128) 1,2,4-Trihydroxynonadecane (118) (E)-1,2,4-Trihydroxynonadec-6-ene (119) Coclaurine (16) N-Methycoclaurine (17) Crostparine (NMS) Glaziovine (18) Caaverine (19) Laurotetanine (20) Nordomesticine (24) Norisoboldine (21) Norantenine (NMS) Corytuberine (22) Domesticine (25) Isoboldine (23) Pallidine (26) NT 12.5b**** 48.6c*** 56c*** 11.5c*** 104c*** 16.5c*** 42b* 20k* 0.4k* 200k* 3241h** 162.5b* NT NT NT NT NT NT NT NT NT NT 183k* 213k* 245k* 276k* 307k* 327k* 189k* 223k* NT NT NT NT NT NT NT NT NT NT NT NT NT

NT NT NT NT NT NT NT NT 18.32b* 15.3b* NT NT NT NT NT 14.2b* NT NT NT NT 264b* NT NT NT 2.2c* 4.4c* 17 407c* 47.3c* 12.6c* 34.8c* 12.2c* 244k* 228k* 198k* 337k* 307k* 294k* 153k* 159k* 10d* 22d* 0d* 42d* 70d* 7d* 76d* 0d* 12d* 0d* 0d* 7d* 8d*

8c# 5.7c# 7
c#

55 55 55 55 55 55 55 55 56 57 58 58 58 58 58 59 60 60 60 61 59 62 62 62 63 63 63 63 63 63 63 33 33 33 33 33 33 33 33 64 64 64 64 64 64 64 64 64 64 64 64 64

4c# 39.2c# 34.7c# 56c# NAc# NT 2b**** 17.4c# 16.6c# 25.9c# ND 35.8c# NT NT NT NT NT NT 75i**** 38i**** 83i**** NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT

Fabaceae Cassia stula Pterodon pubescens Myrospermum frutescens

Gramineae Cymbopogon citratus Lamiaceae Dracocephalum komarovi Salvia gilliessi Ocimum basilicum Lauraceae Laurus nobilis Nectandra megapotamica

Persea americana

Ocotea lancifolia (bark)

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Table 1 (Contd. ) Activity in T. cruzia Species Lecythidaceae Bertholletia excelsa (bark) Meliaceae Pseudocedrela kotschyi Compounds Epimastigotes Trypomastigotes 42.41d* Intracellular amastigotes Ref.

Betulinic acid (108)

NT

NT

65

7-Deacetylgedunin (87) Kotschyin A (88) 7-Deacetyl-7-oxogedunin (89) Eugenol (131) 3-Heptadecyl-5-methoxyphenol (133) Embelin (134) Eupomatenoid-3 (69) Eupomatenoid-5 (70) Eupomatenoid-6 (71) Conocarpan (72) Grandisin (73) rel-(7R,8R,70 R,80 R)-30 ,40 -Methylenedioxy-3,4,5,50 tetramethoxy-7,70 -epoxylignan (74) rel-(7R,8R,70 R,80 R)-3,4,30 ,40 -Dimethylenedioxy-5,50 dimethoxy-7,70 -epoxylignan (75) Isonotholaenic acid (132) 4-Methoxy-6-[2-(methylamino)phenyl]-2H-pyran-2-one (27) rel-(7R,8R)-8-[(E)-3-Hydroxy-3-methyl-1-butenyl]-4,8dimethoxy-5,6,7,8-tetrahydrofuro[2,3-b]quinoline-7-yl acetate (28) Arborinine (29) N-Methyl-1-hydroxy-3-methoxyacridone (30) Skimmianine (31) Kokusagine (32) Canthin-6-one (33) 5-Methoxycanthin-6-one (34) Canthin-6-one N-oxide (35) Sarachine (36) Catechin (54) Epicatechin (55) Gallocatechin (56) Epigallocatechin (57) Catechin gallate (58) Epicatechin gallate (59) Gallocatechin gallate (60) Epigallocatechin gallate (61)

NT NT NT 246b* NT NT 26.3b**** 7b**** 7.5b**** 8b**** NT NT NT

NT NT NT 76b* 50e* 100e* NT NT NT NT 8.74b* 17.6b* 3.47b*

5.2b**** >30b**** >30b**** NT NT NT NT 5b** NT NT NT NT NT

66 66 66 59 67 67 68 68,69 68 68 70 70 70

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Myrtaceae Syzygium aromaticum Oxalidaceae Oxalis erythrorhiza (aerial parts) Piperaceae Piper regnellii (leaves)

Piper solmsianum (owers)

Polypodiaceae Notholaena nivea Rutaceae Almeidea rubra (leaves)

50b*** NT NT NT NT NT NT NT NT NT 10b** 0.000067c* 0.000085c* 0.0000105c* 0.000013c* 0.000048c* 0.000056c* 0.00000012c* 0.00000053c*

>100b* 1271c* 977c* 1231c* 2600c* 1455c* 559c* Dg Dg Dg NT NT NT NT NT NT NT NT NT

NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT

30 71 71 71 71 71 71 72 72 72 73 74 74 74 74 74 74 74 74

Zanthoxylum chiloperone (bark) Solanaceae Saracha punctata Theaceae Camellia sinensis

NA: no activity detected, NT: not tested, NMS: no molecular structure provided, D: decrease in parasitemia. Time of incubation: * 24 h, **48 h, *** 72 h, **** 96 h, # 168 h. b Concentration in mg mL1 able to cause lysis or inhibit growth of 50% of the cells. c Concentration in mM able to cause lysis or inhibit growth of 50% of the cells. d Percentage of lysis or inhibition of growth in the presence of 250 mg mL1 of the compound. e Percentage of lysis or inhibition of growth in the presence of 100 mg mL1 of the compound. f Concentration in mg mL1 able to cause death/ lysis of 100% of the cells. g Parasitemia in animal model. h Percentage of death/lysis in 4.5 mM of the compound. i Percentage of inhibition of growth in 1 mg mL1 of the compound. j Percentage of inhibition of growth in 4 mg mL1 of the compound. k Concentration in mM able to cause death/lysis of 100% of the cells.

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from the Lauraceae showed moderate activity against trypomastigotes. Compounds 19 and 24 caused lysis of 70% and 76% of parasites, respectively; the only difference between these two compounds is the presence of a dioxole ring on 24. Comparing compounds 24 and 25, the presence of a methyl bound to nitrogen on 25 caused inactivation.
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The in vivo evaluation of b-carboline alkaloids indicated decreased parasitemia. Compound 32, a quinoline alkaloid, had moderate activity compared to 28 and 31, which have hydrogenated carbons and methoxy groups in place of a dioxole ring. Protoberberine, quinoline, acridine, and other alkaloids showed poor activity against trypomastigotes. Compound 36, a steroidal
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alkaloid, was tested against epimastigotes and had an IC50 of 10 mg mL1 after 48 h. Flavonoids (3761) are the second most abundant group of isolated compounds, represented here by the families Asteraceae, Bignoniaceae, Clusiaceae, Fabaceae, and Theaceae. Among avonoids evaluated against trypomastigotes, structures 37 and 53 presented the best activity, with approximately 20 mg mL1 needed to cause 50% parasite lysis. Both compounds have the same functional groups bound to the rings, but 53 is an

isoavonoid in which the aromatic ring is bound to the carbon closest to the carbonyl. Comparing glucosylated avonoids, compound 48, glucosylated at the A ring and without methoxy groups, was more active than both 46 and 47, which are both glucosylated on the C ring and have a methoxy on the B ring. Compound 49 was inactive at the concentration tested and differs from 40, which is active, in that it has a methoxy group in the A ring instead of a hydroxy group. The presence of methoxy groups in the B or C rings reduces signicantly the trypanocidal

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activity of these avonoids. In contrast, 43, with a methoxy group in the A ring and a double bond in the C ring, had a considerable lytic effect on the parasite. Compounds 44 and 45, with unsaturated C rings and dihydroxylated B rings, had similar activity; however, 45 lacks a methoxy group in the C ring and was less effective than 44. Several catechins, such as compound 60, for which the IC50 was 0.00000012 mM after 24 h, have shown good activity against epimastigotes. Among catechins, compounds trihydroxylated in the A ring and also trihydroxylated in the B ring were more active against T. cruzi than other catechins. Biavonoids were evaluated for activity only against amastigotes. Compound 52 is dihydroxylated in the B ring and was the only inactive compound of this type. Against the intracellular form, compound 50 inhibited 50% of parasite replication at 34.7 mM after 168 h. Compounds 50 and 51 were effective in different concentrations, with the former being more toxic to the parasites and presenting in its structure a methoxy group in the B ring of the inferior avonoid.

Lignans (6275) were isolated from the families Lauraceae and Piperaceae, with all compounds obtained from the latter family being lignans. In general, lignans showed good activity against all T. cruzi forms. Among tetrahydrofuran lignans from the family Lauraceae, compounds 62 and 63 eliminated 50% of trypomastigotes within 24 h at concentrations of 2.2 and 4.4 mM, respectively. Other good activities against the non-replicative form of the parasite were shown by 66 and 68, with the two being toxic at almost the same concentration of approximately 12 mM. Comparing 63 and 68, we nd that a change in stereoconguration is responsible for the 3-fold higher concentration of drug necessary to exhibit the same activity. Also, the only difference between 64 (which showed no effect) and 67 is the conguration of a methyl group. From the family Piperaceae, compounds were active at low concentrations, and 75 was the best, eliminating 50% of trypomastigotes at 3.47 mg mL1. The presence of two dioxole rings seems to increase the antiparasitic effect for these

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tetrahydrofuran lignans. Neolignans were toxic to epimastigotes, of which 70, 71, and 72 were active at similar concentrations ranging from 7 to 8 mg mL1 after 96 h. In this case, the dioxole ring of 69 in the place of methoxy and hydroxy groups may be the cause for the reduction of activity. The only lignan assayed against amastigotes was 70, and this compound inhibited intracellular replication at 5 mg mL1 after 48 h. Diterpenes (7689) have been isolated from members of the families Meliaceae, Asteraceae, Fabaceae, and Lamiaceae. Only compound 77, of all 136 listed in Table 2, has been evaluated against all forms of the parasite. It showed 50% inhibition of epimastigotes and amastigotes at 12.5 and 2 mg mL1, respectively, after 96 h. Against trypomastigotes, compound 77 had a 50% effect at 15.3 mg mL1 after 24 h. Among other diterpenes from the family Fabaceae, 80 presented good activity together with 82 in epimastigotes, but compound 81 had an IC50 above 100 mM. Independent of the existence of a double bond or a hydroxy group, the carboxy group could be reducing the activity of the latter. Compound 84 from the family Lamiaceae also showed strong activity against epimastigotes, with 0.4 mM causing total lysis of the parasite after 24 h. Diterpenes from the family Meliaceae were screened against amastigote forms of T. cruzi. Compound 87, another diterpene with good activity against intracellular forms, had an IC50 of 5.2 mg mL1 after 96 h. Compounds 88 and 89 showed no activity except at the maximum concentration tested. Comparing 87 and 89, the hydroxy group in the former (compared to the carbonyl group in the latter) is responsible for the activity detected. Sesquiterpenes (90102) were obtained from members of the families Asteraceae and Lauraceae, and all isolated compounds are sesquiterpene lactones. The activity against T. cruzi was better for both replicative forms than for the non-replicative form. From the Asteraceae, sesquiterpenes were assayed against amastigotes, and 91 was the best, inhibiting the parasite inside host cells at 5.72 mg mL1. Compounds 90 and 92 presented moderate and low activity, respectively. For those sesquiterpenes, an acetoxy group in the place of a hydroxy group inactivated the compound. Against epimastigotes, compound 97 exhibited an IC50 of 0.5 mg mL1 after 96 h, while for amastigotes, 91 showed 50% inhibition at 5.3 mg mL1 for the same incubation time. Compound 96 was evaluated at 250 mg mL1 against trypomastigotes, and after 24 h, 98.9% of the parasites were lysed.

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Monoterpenes (103105) were isolated from members of families Gramineae, Lamiaceae, and Lauraceae, while triterpenes (106108) were obtained from species of the Lecythidaceae and Bignoniaceae families. For epimastigotes, 103 showed 50% parasite-inhibiting activity at 42 mg mL1 after 24 h; it had better activity against trypomastigotes, with 14.2 mg mL1 causing 50% lysis. Compound 106 had low activity against trypomastigotes after 24 h, with 400 mg mL1 being necessary to achieve 100% lysis. In comparison, the concentration of 107 to achieve the same results was approximately 4-fold higher.

Xanthones (109113) were obtained from the family Clusiaceae, and anthraquinones (114117) from the family Asphodelaceae. The compounds from both groups have been evaluated
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only against the intracellular form of the parasite. Compounds 110 and 112 inhibited 50% of amastigote growth after 168 h at 5.7 and 4 mM, respectively, while compounds 114 and 115 showed IC50 values of 7.6 and 6.8 mg mL1 after 96 h, respectively. Comparing 112 with 113, the latter was almost 10 times less active due to a differently stereocongured hydrogen. Compounds 114 and 115 had similar activity despite containing different substituents. However, 116 was three times more active than 117, due to the switched positions of the methoxy and hydroxy groups. Oxygenated hydrocarbons 118128 were isolated from members of the Araliaceae and Lauraceae, while aromatic compounds 129134, including phenylpropanoids, were isolated from members of the Myrtaceae, Oxalidaceae, and Polypodiaceae. Compounds 118 and 119, which differ from each other in saturation at position 6, showed similar antiparasitic activities. Against epimastigotes, 123 exhibited an IC50 of 82 mM after 24 h, whereas against trypomastigotes, its activity was moderate, with 198 mg mL1 causing 100% lysis after 24 h. Against amastigotes, 121 and 122 showed IC50 values of 0.2 and 0.15 mg mL1, respectively, after 96 h, which is the best result obtained for oxygenated hydrocarbons. Compounds 120 and 121 differ from each other by the presence of a double bond at position 1 in compound 121, the activity of which is nearly 40fold higher. For compound 123, an acetoxy group in position 4 and a double bond at position 16 promoted high activity. Of the other aromatic compounds, none has been tested against amastigotes, but compounds 131 and 132 showed moderate activity against epimastigotes and trypomastigotes, with 50% lysis detected at doses above 50 mg mL1 in both cases.

In research on Chagas disease, natural products are most often active against the epimastigote form of the parasite. This form is more sensitive to drugs, and is an easy model for the screening of new plant derivatives. The cell structures present in this form differ from the non-replicative trypomastigote and the replicative intracellular amastigote. In particular, epimastigotes have reservosomes; these contain cruzipain, a cysteine protease that is important for the process of differentiation into an infective form and for invasion of host cells.75 An evaluation of reservosomes and their contents, as well as of other proteases, using specic inhibitors in order to determine their effects on the differentiation process may show if enzymatic alteration is occurring. Organelles, including acidocalcisomes, glycosomes, kinetoplasts, and contractile vacuoles, may be affected by the presence of some plant compounds. The fact that these structures are not
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present in the host cells allows them to serve as targets for the development of new drugs.76 However, the actual functions of many parasitic cell structures are still unexplained, which makes the goal of nding new compounds able to alter or regulate the parasite life cycle by affecting these structures a difcult proposition at best. Tests are often not carried out with trypomastigote and amastigote forms of the parasite because many laboratories do not have the nancial resources required to maintain infected animal models or cell cultures. As a result, many compounds are screened only against non-infective forms. Even those that show good results are not studied thoroughly, and often no active compound is isolated. Trypomastigotes can easily evade immune mechanisms and persist in the blood. Even fewer compounds are capable of killing the parasite inside the host, and their effects on the immune response should also be evaluated. Natural products inuence the immune system, and their effects can be manipulated to produce a greater or lesser immune response.77,78 An evaluation of immune responses upon exposure to plant compounds could provide answers about how isolated plant compounds function inside the host. Because the parasite is capable of overcoming the hosts defenses, it is not desirable to discover or employ compounds that interfere with immune responses and, thus, increase host susceptibility to the disease. The amastigote form is the most difcult to affect because its natural habitat is inside host cells, which have many mechanisms for protecting the cell against cytotoxic agents. Compounds must be able to pass through the plasma membrane of the host, and therefore, substances that increase its permeability or act on the efux pumps must be considered. Compounds active against amastigote parasites in cell monolayers have been obtained from plants and were able to reduce the proliferation of the parasite.62 The activity of natural compounds in multilayer tissues could be investigated further by using animal models that would enable evaluation of the activity of various compounds in conditions more closely reecting actual Chagas disease. Just as some compounds affect metabolic pathways by inactivating enzymes or altering the structure of other proteins, it is also possible for a compound to cause overexpression of specic genes. Several alterations in gene expression could be detected in the cell after exposure to various extracts or compounds, thus opening a new avenue for attacking the parasite in a specic manner. Observing nuclear alterations, membrane disorganisation, organelle deformation, or simple changes in the external structures of the parasite would provide more information about the target of the drug.

3 Opportunities for future research

Studying natural substances has the potential to provide benecial results, especially against diseases for which a cure does not yet exist or for which the available treatment is difcult for the patient. Determining the efcacy of a tested drug or its optimum concentration is very important, but providing information about the target of the drug, the biology of the parasite, or its interaction with the host cell is also a valuable additional step.79 Fixed parameters such as the percent efcacy of the drug and time of treatment should be utilised more frequently, as should
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standardisation, to enable better comparisons among the activities of extracts and compounds. An index of 50% activity in parasite lysis, for example, is more understandable as a measure of compound efcacy than merely evaluating the activity of a given compound at the same concentration as the standard drug. In some cases, even if the best activity of an extract or compound is just ten times below the standard drug value, no activity might be observed if only one or two concentrations are analysed. It is necessary to generate activity curves for screening tests with which the percentage of inhibition/lysis can be compared to several concentrations of the drug and used to determine the concentration for 50% activity as a reference datum, and perhaps the concentrations for 90% or 100% activity as well. It is also important to use a standard treatment period, i.e., to expose the parasite and host cells to the drug for the same time period and then to transform the data obtained into a more accurate selectivity index. To expose the cell and the parasite to different incubation periods may lead to an erroneous interpretation of the compounds toxicity because each cell type will require different time periods for detoxication processes and the replacement of damaged structures. In vivo studies always correlate the weight of the drug with the weight of the animal, usually given as mg kg1, and arrive at an agreement as to how the drugs are to be dosed in humans, thus facilitating the determination of the toxic dose. In vitro studies may show a similar dosage but correlate the weight of the drug with the volume of the solvent and not simply the molecular weight of the compound. When a drug is reported in mg mL1 or mg mL1, for example, it is easier to appreciate an increase or decrease in the activity of the drug, and it would help many researchers who study the same family or genus to know the prospects for each one. The molecular weights of these compounds should be available for reference, but many publications do not provide this information. To provide only the name of the compound or its molecular structure and then show the results only in terms of weight makes it difcult to compare the evaluated compounds, because this obliges the reader to calculate the molecular weight in order to determine which compound shows the best activity. Thus a large amount of time is taken to reach a conclusion that could easily be given by the writer. More information on the activity of natural products by the authors of such studies would facilitate additional comparative studies and lead to more rapid advances in research on protozoal diseases. Differences among parasitic protozoa such as Trypanosoma sp., Leishmania sp., Plasmodium sp., and Trichomonas vaginalis are well known. Some investigators have demonstrated the effects of several plant extracts against these protozoa.34,42,64,67,80,81,82 The results are interesting not only because of the high activity of some extracts, but also because they have similar effects on protozoa from different orders and families. Might this similar activity be related to similar targets? What do these parasites have in common that makes them susceptible to the same drug? What experiments can we design to explore these ideas? There still is a black hole at the center of our knowledge of protozoal diseases. Part of this is due to the problem of lack of easy coordination and comparison of information, which could
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be solved if many authors analysed their results better and crossreferenced them with others. This globalisation of scientic data must continue to enable research to progress and to avoid repeating the same steps. Isolation and purication of natural compounds is neither an easy nor a simple process. Extractions have been carried out with hundreds of plant species, and only 136 compounds have so far been obtained, as described above. The fact that so few compounds have been studied is itself the source of another problem, namely that only a few of these have been evaluated against different strains or different protozoa.61 At the very least, the determination of the major compounds in the extracts can help to answer questions about the biological activity of the species. Toxicity is the major impediment in the development of new drugs for parasitic diseases. The treatments for Chagas disease, leishmaniasis, sleeping sickness, malaria, and toxoplasmosis, for example, all have serious side-effects on the hosts. In the case of Chagas disease, a low rate of cure is achieved after the establishment of the chronic phase, and the treatment itself causes the patient to suffer as a consequence of the toxicity. Only 37% of the articles published in the last decade reported results for the toxicity of the extracts or compounds they studied. The differences between our cells and those of parasites, and the discovery of natural products able to act specically on one type of cell, are topics that should be studied in the near future, especially when improved methods for the screening of plants are available. The determinations of lethal doses in animals and investigations of toxicity to mammalian cell lines can help us to understand and address the toxic effects of natural products, thus making it possible to determine whether a potential drug can be used in the same form in which it was isolated, or whether it requires structural modications. Experiments with active compounds isolated from plants have shown that some modications in the molecule increase activity signicantly; however, such alterations of the original compound are rarely performed, and opportunities to nd new drugs are perhaps being missed.83 Synergetic effects on T. cruzi exposed to multiple compounds have mostly been studied using synthetic drugs, and almost no results for natural products used together have been published. Recently, a synergistic effect on T. cruzi epimastigotes was demonstrated between parthenolide, a sesquiterpene lactone isolated from Tanacetum vulgare (Asteraceae), and benznidazole; the IC50 of benznidazole was reduced 23-fold when combined with the natural compound.84 Against Plasmodium, this association of natural compounds has been studied, resulting in the detection of moderate synergism.85 Associations of compounds are already used to treat some diseases such as malaria and sleeping sickness, especially when treatment with the standard drug fails. The possibility of nding natural compounds or derivatives able to replace the usual drug, or ones that can be used in combination with other drugs, should be taken more seriously. One of the most popular means of treatment with medicinal plants is the preparation and ingestion of teas. This ancient tradition includes recipes in which different plant parts are mixed in certain concentrations and sometimes taken in peculiar ways to obtain the desired result. This suggests that, from the beginning, humans have known of the advantages of mixing
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products from Nature to cure their ills. Current scientic practice is to subdivide or simplify the object of study to discover its minimally relevant or functional part, and this also occurs with studies of natural products. However, we must not neglect to put all these minimal parts together again so as to understand the complexity of Nature on the wider scale, where things work together and not separately.

Acknowledgements

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We thank Andr Luis Rdiger for excellent technical assistance. e u This study was supported through grants from the Conselho Nacional de Desenvolvimento Cient co e Tecnolgico CNPq, o Capacitacao de Aperfeicoamento de Pessoal de N Superior vel CAPES, Financiadora de Estudos e Projetos FINEP, PRONEX/Fundacao Araucria, INCT_if, and Programa de Ps a o graduacao em Microbiologia da Universidade Estadual de Londrina.

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